Directional cell migration is essential for organism development, immune surveillance and wound repair, but is also associated with numerous pathologies including cardiovascular and neurodegenerative disorders as well as tumor metastasis. Paxillin is a scaffold protein that localizes to sites of cell-matrix interaction calle focal adhesions and we have previously shown that paxillin functions primarily as a hub for regulating Rho GTPase signaling to coordinate actin cytoskeleton remodeling and focal adhesion turnover to drive cell migration. Importantly, polarized trafficking of pro-migratory factors including integrins, through the secretory machinery to the cell's leading edge, is also required for efficient directional migration. However, the mechanism by which cell motility and polarized trafficking are integrated is not well understood. In this continuing application, we wil evaluate a novel role for paxillin as a fundamental regulator of the cell polarization machinery, through its ability to modulate microtubule function via control of alpha-tubulin acetylation. Specifically, we will use a combination of RNAi and mutant expression, biochemical assays, combined with fixed and real-time fluorescence imaging, to test the hypothesis that paxillin regulates microtubule acetylation via the deacetylase HDAC6 to control Golgi organization and positioning, as well as ER-Golgi-plasma membrane trafficking of proteins including integrins and the metalloproteinase MT1-MMP to regulate directional cell migration and invasion. We will examine the influence of environmental context on this signaling axis by comparing 2D with 1D/3D matrix models of cell polarity. Furthermore, these studies will be performed in both normal and cancer cells in order to identify ubiquitous versus cancer specific roles for paxillin signaling through HDAC6 in polarized trafficking and migration that, in turn, may facilitate the development of therapeutic intervention strategies for various cell migration-related disorders.